Circadian clocks control a wide variety of fundamental cellular, physiological, and behavioral processes in eukaryotic organisms. The molecular machinery that permits the measurement of time is referred to as the circadian clock and its output as circadian rhythms. Our long-term goal is to understand the molecular and biochemical mechanisms of circadian clocks. The filamentous fungus Neurospora crassa, which has one of the best understood circadian clock systems, offers a powerful experimentally-accessible system for exploring the clock mechanism at molecular levels. Like circadian oscillators in the higher eukaryotic organisms, the Neurospora oscillator consists of an autoregulatory negative feedback loop. In this negative feedback loop, two WHITE COLLAR proteins (WC-1 and WC-2) are the positive elements that form a WC complex that activate the transcription of the frequency (frq) gene. The negative elements are FRQ and FRH, a FRQ-interacting RNA helicase, which form a complex that inhibits the WCC activity. In this proposal, we aim to address several fundamental questions of the clock mechanism. In Specific Aim 1, we will determine how the FRQ-dependent WC phosphorylation regulates its localization and activity. This study will establish the biochemical mechanism for the circadian negative feedback process in Neurospora. We recently discovered a novel post-transcriptional negative feedback loop in which FFC and exosome regulate circadian expression of frq by controlling its RNA degradation. In Specific Aim 2, we will determine the post-transcriptional mechanism that regulates circadian gene expression. This study will establish the functional importance of an important post-transcriptional process that controls circadian gene expression. In Specific Aim 3, we will determine the circadian function of a recently identified clock gene that regulates circadian period length and amplitude, and we will clone two additional clock genes by genetics and deep sequencing methods. The identification of new clock genes and their characterization will yield novel and important mechanistic insights into clock mechanisms. These related but independent objectives will help us to elucidate the mechanism of the Neurospora clock in genetic, biochemical, and molecular terms. Because of the similarities between the Neurospora and animal clocks, our results will provide important information on how eukaryotic circadian clocks function.